Glycosylated Beta-Galactosidase Compositions Having improved Transgalactosylating Activity

ABSTRACT

The present invention relates to compositions, particularly liquid compositions, comprising polypeptides having beta-galactosidase activity, methods of making said compositions, and uses of the compositions for making e.g. dairy products. The polypeptides having beta-galactosidase activity are modified by glycation of lysine and/or arginine residues by incubating the enzyme in the presence of reducing sugars, optionally combined with a heat treatment. Thereby, transgalactosylating activity is increased.

SEQUENCE LISTING

The present invention comprises a sequence listing, which isincorporated by reference herein.

TECHNICAL FIELD

The present invention relates to compositions, particularly liquidcompositions, comprising enzymes, methods of making the compositions,and uses of the same for making e.g. dairy products.

BACKGROUND OF THE INVENTION

Beta-galactosidase, also known as lactase, is an enzyme known tohydrolyse the terminal non-reducing beta-D-galactose residues inbeta-D-galactosidases. More particularly, under normal reactionconditions, the enzyme hydrolyses its lactose substrate to the componentmonosaccharides D-glucose and D-galactose. Under certain conditions,certain beta-galactosidases have the ability to transfer galactose tothe hydroxyl group of either glucose or galactose to formgalacto-oligosaccharides (GOS) in a process called transgalactosylation.

A lactase from Bifidobacterium bifidum has been described having a hightransgalactosylating activity, both in the full-length form andespecially when truncated from the C-terminal end (see, e.g., Jørgensenet al. (2001), Appl. Microbiol. Biotechnol., 57: 647-652 or EP patent 1283 876).

In WO 2009/071539, we describe a differently truncated fragment comparedto Jørgensen. WO 2009/071539 discloses C-terminally truncated fragmentof the extracellular lactase from Bifidobacterium bifidum, which wasoriginally isolated and patented for its ability to make high amounts ofgalactooligosaccharides from lactose, can be used very successfully forhydrolysis of lactose in milk. When tested in water+100 g/l lactose at37° C., the enzyme makes galactooligosaccharides with high efficiency asdescribed in the prior art. However, when tested in milk, the ratio ofhydrolytic to transgalactosylating activity has changed markedly,resulting in efficient hydrolysis and very low production ofgalactooligosaccharides.

WO 2013/182686 describes still further differently truncated fragmentscompared to Jørgensen, described as efficient producers of GOS whenincubated with lactose even at low lactose levels such as in amilk-based product. WO 2013/182686 also describes compositionscomprising a stabilizer.

WO 2015/132349 describes liquid lactase compositions comprising lactaseand further comprising sodium, calcium or potassium-L-lactate or acombination thereof and optionally a sugar.

There remains a need to develop enzymes which are efficient producers ofGOS, and industrially important formulations of the same.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a formulation comprising apolypeptide having beta-galactosidase activity and at least 30 wt % of areducing sugar, preferably fructose, galactose, glucose, or lactose.

In another embodiment, the invention provides a polypeptide havingbeta-galactosidase activity having been modified by glycation of atleast one lysine and/or arginine residue.

In another embodiment, the invention provides a method of modifying apolypeptide having beta-galactosidase activity comprising contacting thepolypeptide with a reducing sugar, preferably fructose, glucose,galactose, or lactose, for a time and temperature sufficient to producea polypeptide modified by glycation.

In another embodiment, the invention provides a method for producinggalacto-oligosaccharides (GOS) comprising contacting a formulation ofthe invention or a polypeptide of the invention or a polypeptide havingbeta-galactosidase activity which has been modified by a method of theinvention with lactose.

In still another embodiment, the invention provides a method forproducing galacto-oligosaccharides comprising contacting a polypeptidehaving a sequence comprising or consisting of amino acids 1-1304 of SEQID NO: 1, with lactose under conditions of high temperature and highinitial lactose concentration.

DETAILED DISCLOSURE OF THE INVENTION

Despite the dominant hydrolytic properties of certain beta-galactosidaseor lactase enzymes, these enzymes can be forced to have transferringproperties at, e.g., high lactose and high temperature conditions. Wehave surprisingly discovered that when subjected to a pre-incubation,the previously hydrolytic-dominating enzyme can be converted to atransferring enzyme, which is also able to make GOS efficiently at lowertemperatures than the unprocessed enzyme. The pre-incubation thussurprisingly results in a more robust GOS-producing enzyme due to itsheightened transferring abilities (transgalactosylase activity).

Without wishing to be bound by theory, it is believed that theseincubation conditions result in glycation of the beta-galactosidase,which results in increased transferring properties. With covalentattachment of the sugar moiety, the beta-galactosidase is converted froma hydrolysing to a transferring enzyme having transgalactosylaseactivity.

Beta-Galactosidase Beta-galactosidases from glycoside hydrolase family 2(GH2) are exo-acting enzymes, which hydrolyse terminal non-reducingbeta-D-galactose residues in beta-D-galactosides, e.g. lactose ishydrolysed to galactose and glucose. They belong to the enzyme class EC3.2.1.23 with the official name beta-D-galactoside galactohydrolase. Acommon name used for this enzyme is lactase, as lactose is the commonindustrial substrate. Besides hydrolysing this enzyme class is also ableto transfer galactose to other sugars and thereby makegalacto-oligosaccharides (GOS). The different GH2 enzyme have variouspreferences for hydrolytic or beta-galactosidase activity andtransgalactosylase activity and the preference can be expressed in termsof their GOS production ability, such as by the ratio oftransgalactosylating activity to beta-galactosidase activity.

In the present context, the term “beta-galactosidase” means anyglycoside hydrolase having the ability to hydrolyse the disaccharidelactose into its constituent galactose and glucose monomers. Enzymesassigned to subclass EC 3.2.1.108, also called lactases, are alsoconsidered a beta-galactosidase in the context of the present invention.In the context of the invention, the lactose hydrolysing activity of thebeta-galactosidase may be referred to as its lactase activity or itsbeta-galactosidase activity.

In the context of the present invention, the polypeptide havingbeta-galactosidase activity preferably belongs to the enzyme class EC3.2.1.23 or EC 3.2.1.108, preferably 3.2.1.23. The polypeptide havingbeta-galactosidase activity preferably belongs to glycoside hydrolasefamily 2 (GH2), more preferably to the glycoside hydrolase family GH2_5.

In certain applications, combinations of polypeptides havingpredominantly transgalactosylating activity and predominantlyhydrolysing activity may be contemplated. This may be especially usefulwhen there is a desire to reduce residual lactose after treatment withthe polypeptide having beta-galactosidase activity, for example at lowlactose levels.

When considering the reaction of the polypeptide in e.g. milk,carbohydrates are initially present in the form of lactose, adisaccharide composed of galactose and glucose that is found in milk. Inthe formation of GOS, successive galactose molecules are added tolactose, and then after prolonged incubation a mixture of the variouscarbohydrates is present (glucose, galactose and ˜30 different di- andpolysaccharides).

The term “disaccharide” as used herein means two monosaccharide unitsbound together by a covalent bond known as a glycosidic linkage formedvia a dehydration reaction, resulting in the loss of a hydrogen atomfrom one monosaccharide and a hydroxyl group from the other. In oneaspect, the disaccharide is cellobiose, fucose, lactose, lactulose,maltose, rhamnose, or sucrose, most preferably lactose.

As used herein, the term “transgalactosylase” means an enzyme that isable to transfer galactose to the hydroxyl groups of D-galactose (Gal)or D-glucose (Glc) whereby galactooligosaccharides are produced. In oneembodiment, transgalactosylase activity is identified by reaction of theenzyme on lactose in which the amount of galactose generated is lessthan the amount of glucose generated at a given time.

More particularly, the transgalactosylase activity or preference for anenzyme to hydrolyze lactose or to produce GOS can be evaluated as theamount of glucose minus galactose generated at any given time duringreaction or by direct quantification of GOS generated during thereaction. This measurement may be performed by one of several waysincluding the methods shown in the Examples herein.

When evaluating the transgalactosylating activity versusbeta-galactosidase activity of an enzyme, the beta-galactosidaseactivity is measured as concentration of galactose generated at any timepoint during the reaction.

In the present context, the GOS production of a polypeptide is measuredas

$\frac{\left( {{Glucose} - {Galactose}} \right)}{Galactose},$

i.e., the ratio of transgalactosylating activity to beta-galactosidaseactivity.

Preferably, the ratio of transgalactosylating activity tobeta-galactosidase activity is at least 1, at least 2.5, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 11, or at least 12 as measured in high lactoseconditions.

Polypeptides having beta-galactosidase activity useful according to thepresent invention may be of animal, of plant or of microbial origin.Preferred polypeptides are obtained from microbial sources, inparticular from a filamentous fungus or yeast, or from a bacterium.

The polypeptide may, e.g., be derived from a strain of Agaricus, e.g. A.bisporus; Ascovaginospora; Aspergillus, e.g. A. niger, A. awamori, A.foetidus, A. japonicus, A. oryzae; Candida; Chaetomium; Chaetotomastia;Dictyostelium, e.g. D. discoideum; Kluveromyces, e.g. K. fragilis, K.lactis; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus;Neurospora, e.g. N. crassa; Rhizomucor, e.g. R. pusillus; Rhizopus, e.g.R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, e.g. S.libertiana; Torula; Torulopsis; Trichophyton, e.g. T. rubrum;Whetzelinia, e.g. W. sclerotiorum; Bacillus, e.g. B. sp. B. coagulans,B. circulans, B. megaterium, B. novalis, B. subtilis, B. pumilus, B.stearothermophilus, B. thuringiensis; Bifidobacterium, e.g. B. animalis,B. bifidum, B. breve, B. infantis, B. lactis, B. longum;Chryseobacterium; Citrobacter, e.g. C. freundii; Clostridium, e.g. C.perfringens; Diplodia, e.g. D. gossypina; Enterobacter, e.g. E.aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, e.g. E.herbicola; Escherichia, e.g. E. coli; Klebsiella, e.g. K. pneumoniae;Miriococcum; Myrothesium; Mucor; Neurospora, e.g. N. crassa; Proteus,e.g. P. vulgaris; Providencia, e.g. P. stuartii; Pycnoporus, e.g.Pycnoporus cinnabarinus, Pycnoporus sanguineus; Ruminococcus, e.g. R.torques; Salmonella, e.g. S. typhimurium; Serratia, e.g. S.liquefasciens, S. marcescens; Shigella, e.g. S. flexneri; Streptomyces,e.g. S. antibioticus, S. castaneoglobisporus, S. violeceoruber;Trametes; Trichoderma, e.g. T. reesei, T. viride; Yersinia, e.g. Y.enterocolitica.

In a preferred embodiment, the polypeptide is a beta-galactosidase froma bacterium, e.g. from the family Bifidobacteriaceae, such as from thegenus Bifidobacterium, such as from a strain of B. animalis, B. bifidum,B. breve, B. infantis, B. lactis, or B. longum. In a more preferredembodiment, the polypeptide is a beta-galactosidase from Bifidobacteriumbifidum.

In a preferred embodiment, the polypeptide is a beta-galactosidase froma bacterium, e.g. from the family Bacillaceae, such as from the genusBacillus, such as from a strain of B. sp. B. coagulans, B. circulans, B.megaterium, B. novalis, B. subtilis, B. pumilus, B. stearothermophilus,B. thuringiensis; Bifidobacterium, e.g. B. animalis, B. bifidum, B.breve, B. infantis, B. lactis, B. longum. In a more preferredembodiment, the polypeptide is a beta-galactosidase from Bacilluscirculans or Bacillus infantis.

A preferred polypeptide is a beta-galactosidase having a sequence whichis at least 50%, such as at least 60%, at least 70%, at least 80%, atleast 90%, at least 95% or at least 98% identical to amino acids 1-1304of SEQ ID NO: 1 or a fragment thereof having beta-galactosidaseactivity. Such fragment of SEQ ID NO: 1 may be any fragment of SEQ IDNO: 1 having beta-galactosidase activity.

In a preferred embodiment, a polypeptide having beta-galactosidaseactivity to be used in a method of the present invention comprises anamino acid sequence which is at least 50% identical to amino acids28-1931 of SEQ ID NO: 2, or a fragment thereof having beta-galactosidaseactivity. In a more preferred embodiment, the enzyme comprises an aminoacid sequence which is at least 60%, such as at least 70%, at least 80%,at least 90%, at least 95% or at least 98% identical to amino acids28-1931 of SEQ ID NO: 2.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to amino acids 28-1331 of SEQID NO: 3, or a fragment thereof having beta-galactosidase activity.Preferably, the polypeptide has an amino acid sequence which is at least60%, such as at least 70%, at least 80%, at least 90%, at least 95% orat least 98% identical to amino acids 28-1331 of SEQ ID NO: 3.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to SEQ ID NO: 4, or a fragmentthereof having beta-galactosidase activity. Preferably, the polypeptidehas an amino acid sequence which is at least 60%, such as at least 70%,at least 80%, at least 90%, at least 95% or at least 98% identical toSEQ ID NO: 4.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to SEQ ID NO: 5, or a fragmentthereof having beta-galactosidase activity. Preferably, the polypeptidehas an amino acid sequence which is at least 60%, such as at least 70%,at least 80%, at least 90%, at least 95% or at least 98% identical toSEQ ID NO: 5.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to SEQ ID NO: 6, or a fragmentthereof having beta-galactosidase activity. Preferably, the polypeptidehas an amino acid sequence which is at least 60%, such as at least 70%,at least 80%, at least 90%, at least 95% or at least 98% identical toSEQ ID NO: 6.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to SEQ ID NO: 7, or a fragmentthereof having beta-galactosidase activity. Preferably, the polypeptidehas an amino acid sequence which is at least 60%, such as at least 70%,at least 80%, at least 90%, at least 95% or at least 98% identical toSEQ ID NO: 7.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to SEQ ID NO: 8, or a fragmentthereof having beta-galactosidase activity. Preferably, the polypeptidehas an amino acid sequence which is at least 60%, such as at least 70%,at least 80%, at least 90%, at least 95% or at least 98% identical toSEQ ID NO: 8.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to SEQ ID NO: 9, or a fragmentthereof having beta-galactosidase activity. Preferably, the polypeptidehas an amino acid sequence which is at least 60%, such as at least 70%,at least 80%, at least 90%, at least 95% or at least 98% identical toSEQ ID NO: 9.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to SEQ ID NO: 10, or a fragmentthereof having beta-galactosidase activity. Preferably, the polypeptidehas an amino acid sequence which is at least 60%, such as at least 70%,at least 80%, at least 90%, at least 95% or at least 98% identical toSEQ ID NO: 10.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to SEQ ID NO: 11, or a fragmentthereof having beta-galactosidase activity. Preferably, the polypeptidehas an amino acid sequence which is at least 60%, such as at least 70%,at least 80%, at least 90%, at least 95% or at least 98% identical toSEQ ID NO: 11.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to SEQ ID NO: 12, or a fragmentthereof having beta-galactosidase activity. Preferably, the polypeptidehas an amino acid sequence which is at least 60%, such as at least 70%,at least 80%, at least 90%, at least 95% or at least 98% identical toSEQ ID NO: 12.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to SEQ ID NO: 13, or a fragmentthereof having beta-galactosidase activity. Preferably, the polypeptidehas an amino acid sequence which is at least 60%, such as at least 70%,at least 80%, at least 90%, at least 95% or at least 98% identical toSEQ ID NO: 13.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to SEQ ID NO: 14, or a fragmentthereof having beta-galactosidase activity. Preferably, the polypeptidehas an amino acid sequence which is at least 60%, such as at least 70%,at least 80%, at least 90%, at least 95% or at least 98% identical toSEQ ID NO: 14.

In another embodiment, a polypeptide having beta-galactosidase activityto be used in a method of the present invention has an amino acidsequence which is at least 50% identical to SEQ ID NO: 15, or a fragmentthereof having beta-galactosidase activity. Preferably, the polypeptidehas an amino acid sequence which is at least 60%, such as at least 70%,at least 80%, at least 90%, at least 95% or at least 98% identical toSEQ ID NO: 15.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined as the output of “longest identity”using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol. Biol. 48: 443-453) as implemented in the Needle program of theEMBOSS package (EMBOSS: The European Molecular Biology Open SoftwareSuite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version6.6.0 or later. The parameters used are a gap open penalty of 10, a gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. In order for the Needle program to report thelongest identity, the -nobrief option must be specified in the commandline. The output of Needle labeled “longest identity” is calculated asfollows: (Identical Residues×100)/(Length of Alignment−Total Number ofGaps in Alignment)

A beta-galactosidase may be extracellular. They may have a signalsequence at their N-terminus, which is cleaved off during secretion.

A polypeptide having beta-galactosidase may be derived from any of thesources mentioned herein. The term “derived” means in this context thatthe polypeptide may have been isolated from an organism where it ispresent natively, i.e. the identity of the amino acid sequence of theenzyme are identical to a native polypeptide. The term “derived” alsomeans that the polypeptides may have been produced recombinantly in ahost organism, the recombinantly produced polypeptide having either anidentity identical to a native polypeptide or having a modified aminoacid sequence, e.g. having one or more amino acids which are deleted,inserted and/or substituted, i.e. a recombinantly produced polypeptidewhich is a mutant and/or a fragment of a native amino acid sequence.Within the meaning of a native polypeptide are included naturalvariants. Furthermore, the term “derived” includes polypeptides producedsynthetically by, e.g., peptide synthesis. The term “derived” alsoencompasses enzymes which have been modified e.g. by glycosylation,phosphorylation etc., whether in vivo or in vitro. With respect torecombinantly produced polypeptide the term “derived from” refers to theidentity of the polypeptide and not the identity of the host organism inwhich it is produced recombinantly.

The polypeptide having beta-galactosidase may be obtained from amicroorganism by use of any suitable technique. For instance, abeta-galactosidase polypeptide preparation may be obtained byfermentation of a suitable microorganism and subsequent isolation of alactase preparation from the resulting fermented broth or microorganismby methods known in the art. The polypeptide having beta-galactosidasemay also be obtained by use of recombinant DNA techniques. Such methodnormally comprises cultivation of a host cell transformed with arecombinant DNA vector comprising a DNA sequence encoding the lactase inquestion and the DNA sequence being operationally linked with anappropriate expression signal such that it is capable of expressing thebeta-galactosidase in a culture medium under conditions permitting theexpression of the polypeptide and recovering the polypeptide from theculture. The DNA sequence may also be incorporated into the genome ofthe host cell. The DNA sequence may be of genomic, cDNA or syntheticorigin or any combinations of these, and may be isolated or synthesizedin accordance with methods known in the art.

A polypeptide having beta-galactosidase may be purified. The term“purified” as used herein covers beta-galactosidase enzyme proteinessentially free from insoluble components from the production organism.The term “purified” also covers beta-galactosidase enzyme proteinessentially free from insoluble components from the native organism fromwhich it is obtained. Preferably, it is also separated from some of thesoluble components of the organism and culture medium from which it isderived. More preferably, it is separated by one or more of the unitoperations: filtration, precipitation, or chromatography.

Accordingly, the polypeptide having beta-galactosidase activity may bepurified, viz. only minor amounts of other proteins being present. Theexpression “other proteins” relate in particular to other enzymes. Theterm “purified” as used herein also refers to removal of othercomponents, particularly other proteins and most particularly otherenzymes present in the cell of origin of the beta-galactosidase. Thepolypeptide having beta-galactosidase may be “substantially pure”, i.e.free from other components from the organism in which it is produced,i.e., e.g., a host organism for recombinantly producedbeta-galactosidase. Preferably, the beta-galactosidase is an at least40% (w/w) pure enzyme protein preparation, more preferably at least 50%,60%, 70%, 80% or even at least 90% pure.

The term polypeptide having beta-galactosidase activity includeswhatever auxiliary compounds may be necessary for the enzyme's catalyticactivity, such as, e.g., an appropriate acceptor or cofactor, which mayor may not be naturally present in the reaction system.

The polypeptide may be in any form suited for the use in question, suchas, e.g., in the form of a dry powder or granulate, a non-dustinggranulate, a liquid, a stabilized liquid, or a protected enzyme.

The polypeptide is added in a suitable amount to achieve the desireddegree of lactose hydrolysis under the chosen reaction conditions. Thepolypeptide may be added at a concentration of between 100 and 15,000LAU(C) per litre milk-based substrate, preferably between 100-10,000LAU(C) per litre milk-based substrate. Additional preferredconcentrations include e.g. 100 LAU(C)/L, 250 LAU(C)/L, 500 LAU(C)/L,750 LAU(C)/L, 1000 LAU(C)/L, 1500 LAU(C)/L, 2000 LAU(C)/L, 5000LAU(C)/L, 6000 LAU(C)/L, 7000 LAU(C)/L, 8000 LAU(C)/L, 9000 LAU(C)/L,10,000 LAU(C)/L, 11,000 LAU(C)/L, 12,000 LAU(C)/L, 13,000 LAU(C)/L,14,000 LAU(C)/L, or 15,000 LAU(C)/L.

The activity in LAU(C) of a specific beta-galactosidase may bedetermined by direct measurement of glucose released from lactose. Theskilled person will know how to determine such activity. Alternatively,the activity may be determined by using the activity assay described inthe Methods and Examples of the present application. Here, the activityis obtained by comparing to a standard curve run with abeta-galactosidase of known activity, and the activity of the unknownsample calculated from this.

The activity in LAU(B) of a specific beta-galactosidase may bedetermined by direct measurement of o-nitrophenyl (ONP) released fromo-nitrophenyl β-D-galactopyranoside (ONPG) in a buffer containing 1.46mg/ml substrate in 0.05 M MES, 1 mM MgSO₄ 7H₂O, 450 mg/L Brij 35 atpH6.5 and 30° C. After 600 seconds incubation, the reaction is stoppedby adding 0.2 M Na₂CO₃ and the released ONP is measured at 405 nm after126 seconds incubation. The skilled person will know how to execute thisassay and determine such activity. Here, the activity is obtained bycomparing to a standard curve run with a lactase of known activity, andthe activity of the unknown sample calculated from this. The lactase ofknown activity may, e.g., be Saphera® obtained from Novozymes NS,Denmark.

The skilled person will know how to determine the lactase activity atdifferent pH and temperature. The lactase activity at different pH andtemperature is preferably determined by using a method as described inthe Examples of the present application.

In one aspect, the polypeptide is a fragment having one or more(several) amino acids deleted from the amino or carboxyl terminal of thepolypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,or 15 wherein the fragment has beta-galactosidase activity. Particularlypreferred are fragments which are carboxy-terminal truncations.

A fragment of beta-galactosidase contains at least 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or1300 amino acid residues.

In one aspect, the beta-galactosidase is as described in WO 2013/182686.

In one aspect, the beta-galactosidase is as described in WO 2015/132349.

In an aspect, the beta-galactosidase includes a polypeptide of SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 and one or morefragments having beta-galactosidase activity, such as at least one, two,three, four, or five fragments.

Glycation

In an embodiment, the polypeptide having beta-galactosidase activity hasbeen modified by glycation.

Without wishing to be bound by theory, it has been surprisingly foundthat glycation of the beta-galactosidase converts the polypeptide from amore hydrolysing to a more transferring enzyme having transgalactosylaseactivity.

“Glycation” as used herein refers to the covalent attachment of acarbohydrate to a protein. Carbohydrate attachment may be via a sidechain of, e.g., arginine, lysine, or N-terminal of the enzyme.Preferably, the carbohydrate attachment is via a side chain of arginineor lysine.

Glycation is sometimes referred to as (non-enzymatic) glycosylation. Inthe context of the present invention, glycosylation and glycation areused interchangeably and glycosylation can be non-enzymatic.

In an embodiment, the polypeptide having beta-galactosidase activity hasbeen modified by glycation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60 residues of the polypeptide.

In an embodiment, the polypeptide having beta-galactosidase activity hasbeen modified by glycation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60 lysine and/or arginine residuesof the polypeptide.

In an embodiment, the polypeptide having beta-galactosidase activity hasbeen modified by glycation of at least 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45% or 50% of the lysine and/or arginineresidues of the polypeptide. In one embodiment, the polypeptide havingbeta-galactosidase activity has been modified by glycation of at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60lysine and/or arginine residues of the polypeptide.

In a preferred embodiment, the polypeptide having beta-galactosidaseactivity is modified by glycation of at least 1%, preferably at least3%, more preferably at least 5%, even more preferably at least 10%, mostpreferably at least 20%, of the lysine and arginine residues of thepolypeptide. For the avoidance of any possible doubt, this means that atleast 1%, preferably at least 3%, more preferably at least 5%, even morepreferably at least 10%, most preferably at least 20%, of the totalnumber of lysine and arginine residues of the polypeptide is modified byglycation.

In another preferred embodiment, a trypsin digest of the polypeptidehaving beta-galactosidase activity would result in a percentage ofglycated trypsin digested peptides of at least 1%, preferably at least3%, more preferably at least 5%, at least 10% or at least 20%.

In an embodiment, incubation under suitable conditions as detailed belowresults in the glycation of some, substantially all, or even all of thesurface residues of lysine and/or arginine. Again without wishing to bebound by theory, it is believed that some, substantially all, or evenall of the glycated residues are located towards the C-terminal end ofthe polypeptide having beta-galactosidase activity.

Incubation Resulting in Glycation

In an embodiment, the invention provides a method of modifying apolypeptide having beta-galactosidase activity comprising contacting thepolypeptide with a sugar for a time and temperature sufficient toproduce a polypeptide modified by glycation.

In an embodiment, the polypeptide is contacted with a solution of 5-90wt % sugar at pH 5-10 for a time of 3-20 hours at a temperature of20-80° C. Preferred sugars are reducing sugars as set forth in moredetail below, and particularly preferred is glucose.

Suitable conditions include contacting the polypeptide with a solutionof 5-90 wt % sugar, such as 30-90 wt %, and in particular 30-70 wt %,e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, or 90% sugar.

Suitable conditions include contacting the polypeptide at a pH in therange of 5-10, such as pH 5-8, e.g., pH 5, pH 5.5, pH 6, pH 6.5, pH 7,pH 7.5, pH 8, pH 8.5, pH 9, pH 9.5, or pH 10.

Suitable conditions include contacting the polypeptide for a time in therange of 3-20 hours, such as in the range of 6-16 hours, e.g., 3 hours,3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours, 13 hours, 13.5 hours,14 hours, 14.5 hours, 15 hours, 15.5 hours, 16 hours, 16.5 hours, 17hours, 17.5 hours, 18 hours, 18.5 hours, 19 hours, 19.5 hours, or 20hours.

Suitable conditions include contacting the polypeptide at a temperaturein the range of 20-80° C., such as in the range of 20-50° C.,alternatively in the range of 40-80° C., in particular, 50-70° C., oralternatively, 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C.,55° C., 60° C., 65° C., 70° C., 75° C., or 80° C.

In a preferred embodiment, the polypeptide having beta-galactosidaseactivity is contacted with a reducing sugar at pH 5-8, preferably pH6-7, for a time of 3-100 hours, preferably 15-80 hours, at a temperatureof 50-80° C., preferably 50-70° C.

The skilled person will know how to adjust the time of the contactingwith the sugar according to the amount of enzyme added and thetemperature. In general, if more enzyme is added, the time of contactingcan be reduced. And in general, if the reaction temperature isincreased, the time of contacting can be reduced. Depending on thestorage conditions of the enzyme after the contacting with the sugar,the glycation process may continue on the shelf. Therefore, if the shelftemperature of the enzyme is relatively high, the time of reaction withthe sugar at a specified high temperature may be reduced since theglycation process will continue during transport and storage of theenzyme before it being used by the end consumer, who may be, e.g., adairy company or a company producing GOS as an ingredient.

In another preferred embodiment, the polypeptide havingbeta-galactosidase activity is contacted with 30-90 wt %, preferably40-65 wt %, of a reducing sugar.

Sugar

The sugar in the beta-galactosidase formulation can includemonosaccharides, disaccharides, or oligosaccharides. Blends of sugarsare also contemplated.

Preferably, the sugar is a reducing sugar. A reducing sugar reacts withan amino acid residue of the beta-galactosidase via the Maillardreaction.

Exemplary reducing sugars include the monosaccharides fructose,galactose, glucose, glyceraldehyde, ribose, xylose. Preferred isfructose, galactose and/or glucose and most particularly fructose and/orglucose.

Other exemplary reducing sugars include disaccharides such ascellobiose, lactose and maltose, preferably lactose and/or maltose. Alsoexemplary are glucose polymers e.g. maltodextrin and glycogen.

The presence of a reducing sugar can be detected by many well-knowntests including the use of Benedict's reagent and/or Tollen's reagent.

Formulation

The formulation according to an embodiment of the invention maycomprises a liquid composition. Liquid compositions are preferred forease of use.

In an alternative embodiment, the formulation comprises a solidcomposition, e.g., a powder ora granulate.

In an embodiment, the formulation or composition according to theinvention comprises a polypeptide having beta-galactosidase activity andat least 30 wt %, 31 wt %, 32 wt %, 33 wt %, 34 wt %, 35 wt %, 36 wt %,37 wt %, 38 wt %, 39 wt %, 40 wt %, 41 wt %, 42 wt %, 43 wt %, 44 wt %,45 wt %, 46 wt %, 47 wt %, 48 wt %, 49 wt %, 50 wt %, 51 wt %, 52 wt %,53 wt %, 54 wt %, 55 wt %, 56 wt %, 57 wt %, 58 wt %, 59 wt %, 60 wt %,61 wt %, 62 wt %, 63 wt %, 64 wt %, 65 wt %, 66 wt %, 67 wt %, 68 wt %,69 wt %, 70 wt %, 71 wt %, 72 wt %, 73 wt %, 74 wt %, 75 wt %, 76 wt %,77 wt %, 78 wt %, 79 wt %, 80 wt %, 81 wt %, 82 wt %, 83 wt %, 84 wt %,85 wt %, 86 wt %, 87 wt %, 88 wt %, 89 wt % or 90 wt % sugar. Preferablysuch a beta-galactosidase composition comprises 200-20,000 LAU(C) per g.

In one suitable formulation, the composition comprises enzymepolypeptide having beta-galactosidase activity and at least 30 wt %, 31wt %, 32 wt %, 33 wt %, 34 wt %, 35 wt %, 36 wt %, 37 wt %, 38 wt %, 39wt %, 40 wt %, 41 wt %, 42 wt %, 43 wt %, 44 wt %, 45 wt %, 46 wt %, 47wt %, 48 wt %, 49 wt %, 50 wt %, 51 wt %, 52 wt %, 53 wt %, 54 wt %, 55wt %, 56 wt %, 57 wt %, 58 wt %, 59 wt %, 60 wt %, 61 wt %, 62 wt %, 63wt %, 64 wt %, 65 wt %, 66 wt %, 67 wt %, 68 wt %, 69 wt %, 70 wt %, 71wt %, 72 wt %, 73 wt %, 74 wt %, 75 wt %, 76 wt %, 77 wt %, 78 wt %, 79wt %, or 80 wt % glucose. Preferably such a beta-galactosidasecomposition comprises 200-20,000 LAU(C) per g.

One suitable beta-galactosidase composition comprises 200-20,000 LAU(C)per g and at least 35 wt %, 36 wt %, 37 wt %, 38 wt %, 39 wt %, 40 wt %,41 wt %, 42 wt %, 43 wt %, 44 wt %, 45 wt %, 46 wt %, 47 wt %, 48 wt %,49 wt %, 50 wt %, 51 wt %, 52 wt %, 53 wt %, 54 wt %, 55 wt %, 56 wt %,57 wt %, 58 wt %, 59 wt %, 60 wt %, 61 wt %, 62 wt %, 63 wt %, 64 wt %or 65 wt % sugar, preferably in the range of 40-80 wt % sugar. Apreferred beta-galactosidase composition comprises 200-20,000 LAU(C) perg and preferably 40 wt %, 60 wt %, or 80 wt % glucose.

In one embodiment, the formulation is a liquid formulation whichcomprises 200-15,000 LAU(C)/g, preferably 500-10,000 LAU(C)/g.

In one embodiment, the formulation is a solid formulation whichcomprises 1,000-20,000 LAU(C)/g, preferably 3,000-15,000 LAU(C)/g.

In an embodiment, the formulation further comprises glycerol.

However, in a preferred embodiment, the formulation is free of, or atleast substantially free of, polyols or diols, such as glycerol and/orsorbitol. The amount of polyol or diol such as glycerol is preferablyless than 40 wt %, less than 30 wt %, less than 25 wt %, less than 20 wt%, less than 15 wt %, less than 10 wt %, most preferably less than 5 wt%. Most preferably the formulation is free of polyol or diol such asglycerol.

In an embodiment, the formulations herein are enzymatically stable.Particularly preferred are enzymatically stable liquid enzymeformulations, and more particularly preferred are enzymatically stableliquid enzyme formulations without using glycerol. Enzymatic stabilityis a measure of the rate at which the activity of the enzyme decreasesover time.

Also preferred are formulations, especially liquid formulations, whichare microbially stable. Microbial stability is a measure of the rate atwhich undesired microorganisms can proliferate and grow in thecomposition.

In an embodiment, the formulation further comprises sodium chloride orpotassium chloride, preferably in the range of 0.01-5 wt %, preferably0.01-3 wt %, more preferably 0.01-2 wt %.

In an embodiment, the formulation further comprises a preservative. Foodgrade preservatives are preferred, of which benzoate, sorbate, methylparaben, and propyl paraben are exemplary.

In an alternative but preferred embodiment, the formulation is free ofpreservatives such as benzoate, sorbate, methyl paraben and/or propylparaben.

Uses

Production of galacto-oligosaccharides is contemplated under both insitu conditions from lactose already present in the milk, as well asunder conditions of high initial lactose concentration (greater than40-50% lactose (w/w)).

In an embodiment, methods for producing galacto-oligosaccharidescomprising contacting a polypeptide having beta-galactosidase activitywith lactose under conditions of high temperature and high initiallactose concentration. In particular, the temperature may be, e.g.,40-80° C., such as 50° C., 60° C., 65° C., 70° C., 75° C., or 80° C.Moreover, the initial lactose concentration may be above 40% (w/w), suchas 40-50% (w/w), 45% (w/w), 50% (w/w), 55% (w/w), 40-60% (w/w) or evenabove 60% (w/w), such as 61% (w/w), 62% (w/w), 63% (w/w), 64% (w/w), 65%(w/w), 66% (w/w), 67% (w/w), 68% (w/w), 69% (w/w), 70% (w/w), 71% (w/w),72% (w/w), 73% (w/w), 74% (w/w), 75% (w/w), or 80% (w/w) lactose.

In an aspect is provided a method for producing a dairy productcomprising treating a milk-based substrate comprising lactose with apolypeptide having beta-galactosidase activity as described herein.Typically, under in situ conditions for applications of a polypeptidehaving beta-galactosidase activity in milk, initial lactoseconcentration is about 3-10% (w/w) lactose e.g., 3, 4, 5, 6, 7, 8, 9, or10% (w/w), most typically about 5% (w/w).

The term “milk”, in the context of the present invention, is to beunderstood as the lacteal secretion obtained by milking any mammal, suchas cows, sheep, goats, buffaloes or camels.

“Milk-based substrate”, in the context of the present invention, may beany raw and/or processed milk material. Useful milk-based substratesinclude, but are not limited to solutions/suspensions of any milk ormilk like products comprising lactose, such as whole or low fat milk,skim milk, buttermilk, reconstituted milk powder, condensed milk,solutions of dried milk, UHT milk, whey, whey permeate, acid whey, orcream.

Preferably, the milk-based substrate is milk or an aqueous solution ofskim milk powder. Milk powder typically has a starting lactoseconcentration of 36-52% (w/w/).

The milk-based substrate may be more concentrated than raw milk.

In one embodiment, the milk-based substrate has a ratio of protein tolactose of at least 0.2, preferably at least 0.3, at least 0.4, at least0.5, at least 0.6 or, most preferably, at least 0.7.

The milk-based substrate may be homogenized and pasteurized according tomethods known in the art.

“Homogenizing” as used herein means intensive mixing to obtain a solublesuspension or emulsion. It may be performed so as to break up the milkfat into smaller sizes so that it no longer separates from the milk.This may be accomplished by forcing the milk at high pressure throughsmall orifices.

“Pasteurizing” as used herein means reducing or eliminating the presenceof live organisms, such as microorganisms, in the milk-based substrate.Preferably, pasteurization is attained by maintaining a specifiedtemperature for a specified period of time. The specified temperature isusually attained by heating. The temperature and duration may beselected in order to kill or inactivate certain bacteria, such asharmful bacteria, and/or to inactivate enzymes in the milk. A rapidcooling step may follow.’ A “dairy product” in the context of thepresent invention may be any food product wherein one of the majorconstituents is milk-based. Preferable, the major constituent ismilk-based. More preferably, the major constituent is a milk-basedsubstrate which has been treated with polypeptide havingbeta-galactosidase activity according to a method of the invention. Inthe context of the present invention “one of the major constituents”means a constituent having a dry matter which constitutes more than 20%,preferably more than 30% or more than 40% of the total dry matter of thedairy product, whereas “the major constituent” means a constituenthaving a dry matter which constitutes more than 50%, preferably morethan 60% or more than 70% of the total dry matter of the dairy product.

A dairy product according to the invention may be, e.g., skim milk, lowfat milk, whole milk, cream, UHT milk, milk having an extended shelflife, a fermented milk product, cheese, yoghurt, butter, dairy spread,butter milk, acidified milk drink, sour cream, whey based drink, icecream, condensed milk, dulce de leche or a flavoured milk drink. A dairyproduct may be manufactured by any method known in the art.

A dairy product may additionally comprise non-milk components, e.g.vegetable components such as, e.g., vegetable oil, vegetable protein,and/or vegetable carbohydrates. Dairy products may also comprise furtheradditives such as, e.g., enzymes, flavouring agents, microbial culturessuch as probiotic cultures, salts, sweeteners, sugars, acids, fruit,fruit juices, or any other component known in the art as a component of,or additive to, a dairy product.

In one embodiment of the invention, one or more milk components and/ormilk fractions account for at least 50% (weight/weight), such as atleast 70%, e.g. at least 80%, preferably at least 90%, of the dairyproduct.

In one embodiment of the invention, one or more milk-based substrateshaving been treated with lactase polypeptide having beta-galactosidaseactivity according to a method of the invention account for at least 50%(weight/weight), such as at least 70%, e.g. at least 80%, preferably atleast 90%, of the dairy product.

In one embodiment of the invention, the dairy product is a dairy productwhich is not enriched by addition of pre-producedgalactooligosaccharides.

In one embodiment of the invention, the enzyme-treated milk-basedsubstrate is not dried before being used as an ingredient in the dairyproduct.

In one embodiment of the invention, the dairy product is ice cream. Inthe present context, ice cream may be any kind of ice cream such as fullfat ice cream, low fat ice cream, or ice cream based on yoghurt or otherfermented milk products. Ice cream may be manufactured by any methodknown in the art.

In one embodiment of the invention, the dairy product is milk orcondensed milk. Condensed milk typically has a lactose concentration of10-20% (w/w), such as 10-16% (w/w), and in some embodiments, 18-18.5%(w/w).

In one preferred embodiment of the invention, the dairy product is UHTmilk. UHT milk in the context of the present invention is milk which hasbeen subjected to a sterilization procedure which is intended to killall microorganisms, including the bacterial spores. UHT (ultra hightemperature) treatment may be, e.g., heat treatment for 30 seconds at130° C., or heat treatment for one second at 145° C.

In one preferred embodiment of the invention, the dairy product is ESLmilk. ESL milk in the context of the present invention is milk which hasan extended shelf life due to microfiltration and/or heat treatment andwhich is able to stay fresh for at least 15 days, preferably for atleast 20 days, on the store shelf at 2-5° C.

In another preferred embodiment of the invention, the dairy product is afermented dairy product, e.g., yoghurt.

A “fermented dairy product” in the context of the present invention isto be understood as any dairy product wherein any type of fermentationforms part of the production process. Examples of fermented dairyproducts are products like yoghurt, buttermilk, creme fraiche, quark andfromage frais. A fermented dairy product may be produced by any methodknown in the art.

“Fermentation” in the method of the present invention means theconversion of carbohydrates into alcohols or acids through the action ofa microorganism. Preferably, fermentation in the method of the presentinvention comprises conversion of lactose to lactic acid.

In the context of the present invention, “microorganism” may include anybacterium or fungus being able to ferment the milk substrate.

The microorganisms used for most fermented milk products are selectedfrom the group of bacteria generally referred to as lactic acidbacteria. As used herein, the term “lactic acid bacterium” designates agram-positive, microaerophilic or anaerobic bacterium, which fermentssugars with the production of acids including lactic acid as thepredominantly produced acid, acetic acid and propionic acid. Theindustrially most useful lactic acid bacteria are found within the order“Lactobacillales” which includes Lactococcus spp., Streptococcus spp.,Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp.,Pediococcus spp., Brevibacterium spp., Enterococcus spp. andPropionibacterium spp. Additionally, lactic acid producing bacteriabelonging to the group of anaerobic bacteria, bifidobacteria, i.e.Bifidobacterium spp., which are frequently used as food cultures aloneor in combination with lactic acid bacteria, are generally included inthe group of lactic acid bacteria.

Lactic acid bacteria are normally supplied to the dairy industry eitheras frozen or freeze-dried cultures for bulk starter propagation or asso-called “Direct Vat Set” (DVS) cultures, intended for directinoculation into a fermentation vessel or vat for the production of afermented dairy product. Such cultures are in general referred to as“starter cultures” or “starters”.

Commonly used starter culture strains of lactic acid bacteria aregenerally divided into mesophilic organisms having optimum growthtemperatures at about 30° C. and thermophilic organisms having optimumgrowth temperatures in the range of about 40 to about 45° C. Typicalorganisms belonging to the mesophilic group include Lactococcus lactis,Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp.cremoris, Pseudoleuconostoc mesenteroides subsp. cremoris, Pediococcuspentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis,Lactobacillus casei subsp. casei and Lactobacillus paracasei subsp.paracasei. Thermophilic lactic acid bacterial species include asexamples Streptococcus thermophilus, Enterococcus faecium, Lactobacillusdelbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillusdelbrueckii subsp. bulgaricus and Lactobacillus acidophilus.

Also the anaerobic bacteria belonging to the genus Bifidobacteriumincluding Bifidobacterium bifidum, Bifidobacterium animalis andBifidobacterium longum are commonly used as dairy starter cultures andare generally included in the group of lactic acid bacteria.Additionally, species of Propionibacteria are used as dairy startercultures, in particular in the manufacture of cheese. Additionally,organisms belonging to the Brevibacterium genus are commonly used asfood starter cultures.

Another group of microbial starter cultures are fungal cultures,including yeast cultures and cultures of filamentous fungi, which areparticularly used in the manufacture of certain types of cheese andbeverage. Examples of fungi include Penicillium roqueforti, Penicilliumcandidum, Geotrichum candidum, Torula kefir, Saccharomyces kefir andSaccharomyces cerevisiae.

In one embodiment of the present invention, the microorganism used forfermentation of the milk-based substrate is Lactobacillus casei or amixture of Streptococcus thermophilus and Lactobacillus delbrueckiisubsp. bulgaricus.

Fermentation processes to be used in a method of the present inventionare well known and the person of skill in the art will know how toselect suitable process conditions, such as temperature, oxygen, amountand characteristics of microorganism/s, additives such as e.g.carbohydrates, flavours, minerals, enzymes, and process time. Obviously,fermentation conditions are selected so as to support the achievement ofthe present invention.

As a result of fermentation, pH of the milk-based substrate will belowered. The pH of a fermented dairy product of the invention may be,e.g., in the range 3.5-6, such as in the range 3.5-5, preferably in therange 3.8-4.8.

In a preferred embodiment, the fermented dairy product is yoghurt.

In one embodiment, is provided a method of using a polypeptide havingbeta-galactosidase activity as described herein, or a cell expressingsuch polypeptide, for producing oligosaccharides. Oligosaccharidesinclude, without limitation, fructo-oligosaccharides,galacto-oligosaccharides, isomalto-oligosaccharides, lactosucrose,malto-oligosaccharides, mannan-oligosaccharides, andxylo-oligosaccharides. Particularly preferred aregalacto-oligosaccharides (GOS).

In an embodiment, oligosaccharides are produced by contactingpolypeptide as described herein with a medium that comprises adisaccharide substrate including, e.g., cellobiose, lactose, lactulose,maltose, rhamnose, sucrose, and trehalose, and incubating underconditions whereby oligosaccharides are produced. The medium comprisinga polypeptide as described herein may be part of a product selected fromthe group consisting of cheese, yoghurt, and other fermented milkproducts as also described more particularly above, as well as dietarysupplements and probiotic comestible products. Alternatively, theoligosaccharides can be recovered and subsequently added to the productof interest before or after its preparation.

Similarly, in an embodiment, oligosaccharides may be produced bycontacting a cell expressing enzyme polypeptide as described herein in amedium that comprises a disaccharide substrate including, e.g.,cellobiose, lactose, lactulose, maltose, rhamnose, sucrose, andtrehalose, and incubating under conditions whereby oligosaccharides areproduced. The cells may be part of a product selected from the groupconsisting of cheese, yoghurt, and other fermented milk products as alsodescribed more particularly above, as well as dietary supplements andprobiotic comestible products. Alternatively, the oligosaccharides canbe recovered and subsequently added to the product of interest before orafter its preparation.

In one aspect, the use of a cell for producing a product selected fromthe group consisting of yoghurt, cheese, fermented milk product, dietarysupplement and probiotic comestible product, is provided.

In one aspect, the polypeptides described herein may be used to preparecheese products and in methods for making the cheese products. Cheeseproducts may be e.g., selected from the group consisting of creamcheese, cottage cheese, and process cheese. By adding polypeptides thecheeses may contain significantly increased levels ofgalactooligosaccharides and reduced levels of lactose. In one aspect,the lactose levels in the final cheese product may be reduced by atleast about 25 percent, preferably at least about 50 percent, and morepreferably at least about 75 percent. The polypeptides may be used toreduce lactose in cheese products to less than about 1 gram per serving,an amount that can be tolerated by most lactose-intolerant individuals.

The cheese products provided herein are nutritionally-enhanced cheeseproducts having increased soluble fiber content, reduced caloriccontent, excellent organoleptic properties, improved texture, andflavour. Further, the polypeptides described herein may reduce theglycemic index of the cheese products because GOS are more slowlyabsorbed than lactose or its hydrolysis products. Finally, thepolypeptides may reduce the cost of production of cheese products,particularly cream cheese products, because GOS surprisingly provideimproved texture to the cream cheese product, thus permitting reduceduse of stabilizers, or by allowing for increased moisture contentwithout syneresis.

In a further aspect, the use of a transgalactosylating polypeptide asdisclosed herein or a cell as disclosed herein, for producinggalacto-oligosaccharides, is provided. In one aspect, the use of atransgalactosylating polypeptide as disclosed herein or a cell asdisclosed herein, for producing galacto-oligosaccharides to be part of aproduct selected from the group consisting of yoghurt, cheese, fermenteddairy products, dietary supplements and probiotic comestible products,is provided. In one aspect, the product is yoghurt, cheese, or fermenteddairy products. In one aspect, the use of a transgalactosylatingpolypeptide as disclosed herein or a cell as disclosed herein, forproducing galacto-oligosaccharides to enhance the growth ofBifidobacterium, is provided. In one aspect, the use of atransgalactosylating polypeptide as disclosed herein or a cell asdisclosed herein, for producing galacto-oligosaccharides to enhance thegrowth of Bifidobacterium in a mixed culture fermentation, is provided.

In one aspect, a process for producing a transgalactosylatingpolypeptide as disclosed herein, comprising culturing a cell asdisclosed herein in a suitable culture medium under conditionspermitting expression of said polypeptide, and recovering the resultingpolypeptide from the culture, is provided. A process for producinggalacto-oligosaccharides, comprising contacting of a polypeptide asdisclosed herein or a cell as disclosed herein with a milk-basedsolution comprising lactose, is provided.

The treatment of milk products with a polypeptide that converts lactoseinto monosaccharides or GOS has several advantages. The products may beconsumed by people with lactose intolerance that would otherwise exhibitsymptoms such as flatulence and diarrhea. Dairy products treated withlactase will also have a higher sweetness than similar untreatedproducts due to the higher perceived sweetness of glucose and galactosecompared to lactose. This effect is particularly interesting forapplications such as yoghurt and ice-cream where high sweetness of theend product is desired and this allows for a net reduction ofcarbohydrates in the consumed product. In ice-cream production, aphenomenon termed sandiness is often seen, where the lactose moleculescrystallizes due to the relative low solubility of the lactose. Whenlactose is converted into monosaccharides or GOS the mouth feeling ofthe ice-cream is much improved over the non-treated products. Thepresence of a sandy feeling due to lactose crystallization can beeliminated and the raw material costs can be decreased by replacement ofskimmed milk powder by when powder. The main effects of the enzymatictreatment are increased sweetness.

Another interesting use of the polypeptides having beta-galactosidaseactivity is in infant, follow-on or toddler formula. Infant formula is amanufactured food designed and marketed for feeding to babies andinfants under 12 months of age, usually prepared for bottle-feeding orcup-feeding from a powder (mixed with water) or a liquid (with orwithout additional water). The most commonly used infant formulaecontain purified cow's milk whey and casein as a protein source, a blendof vegetable oils as a fat source, lactose as a carbohydrate source, avitamin-mineral mix, and other ingredients.

In many countries, the addition or carry-over of glycerol to infant,follow-on or toddler formula is prohibited by law, therefore inapplications for infant, follow-on or toddler formula, formulations ofpolypeptides having beta-galactosidase activity must be free ofglycerol.

In one embodiment, the polypeptides having transgalactosylating activitymay be used together with other enzymes such as proteases, includingchymosin or rennin, lipases such as phospholipases, amylases, andtransferases.

PREFERRED EMBODIMENTS

-   -   1. A formulation comprising a polypeptide having        beta-galactosidase activity and at least 30 wt % of a reducing        sugar, preferably fructose, galactose, glucose, or lactose.    -   2. The formulation of embodiment 1, wherein the polypeptide        having beta-galactosidase activity has been modified by        glycation of at least one lysine and/or arginine residue.    -   3. The formulation of any of the preceding embodiments, wherein        the polypeptide having beta-galactosidase activity has been        modified by glycation of at least 1%, preferably at least 3%,        more preferably at least 5%, even more preferably at least 10%,        most preferably at least 20%, of the lysine and arginine        residues of the polypeptide.    -   4. The formulation of any of the preceding embodiments, which is        an enzyme formulation.    -   5. The formulation of any of the preceding embodiments having an        activity of 200-20,000 LAU(C)/g, preferably 500-15,000 LAU(C)/g.    -   6. The formulation of any of the preceding embodiments which is        a liquid formulation, preferably having an activity of        200-15,000 LAU(C)/g, more preferably 500-10,000 LAU (C)/g.    -   7. The formulation of any of the preceding embodiments which is        a solid formulation, preferably having an activity of        1,000-20,000 LAU(C)/g, more preferably 3,000-15,000 LAU(C)/g.    -   8. The formulation of any of the preceding embodiments,        comprising 40-65 wt % sugar.    -   9. The formulation of any of the preceding embodiments, wherein        the sugar is glucose.    -   10. The formulation of any of the preceding embodiments, which        is substantially free of glycerol.    -   11. The formulation of any of the preceding embodiments, which        further comprises sodium chloride or potassium chloride,        preferably in the range of 0.01-5 wt %, preferably 0.01-3 wt %,        more preferably 0.01-2 wt %.    -   12. The formulation of any of the preceding embodiments, wherein        the polypeptide having beta-galactosidase activity has an amino        acid sequence which is at least 50%, such as at least 60%, at        least 70%, at least 80%, at least 90%, at least 95% or at least        98% identical to amino acids 1-1304 of SEQ ID NO: 1 or a        fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, at least 70%, at least 80%,        at least 90%, at least 95% or at least 98% identical to amino        acids 28-1931 of SEQ ID NO: 2, or a fragment thereof having        beta-galactosidase activity; at least 50% identical, such as at        least 60%, such as at least 70%, at least 80%, at least 90%, at        least 95% or at least 98% identical to amino acids 28-1331 of        SEQ ID NO: 3, or a fragment thereof having beta-galactosidase        activity; at least 50% identical, such as at least 60%, such as        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to SEQ ID NO: 4, or a fragment thereof        having beta-galactosidase activity; at least 50% identical, such        as at least 60%, such as at least 70%, at least 80%, at least        90%, at least 95% or at least 98% identical to SEQ ID NO: 5, or        a fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, such as at least 70%, at        least 80%, at least 90%, at least 95% or at least 98% identical        to SEQ ID NO: 6, or a fragment thereof having beta-galactosidase        activity; at least 50% identical, such as at least 60%, such as        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to SEQ ID NO: 7, or a fragment thereof        having beta-galactosidase activity; at least 50% identical, such        as at least 60%, such as at least 70%, at least 80%, at least        90%, at least 95% or at least 98% identical to SEQ ID NO: 8, or        a fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, such as at least 70%, at        least 80%, at least 90%, at least 95% or at least 98% identical        to SEQ ID NO: 9, or a fragment thereof having beta-galactosidase        activity; at least 50% identical, such as at least 60%, such as        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to SEQ ID NO: 10, or a fragment thereof        having beta-galactosidase activity; at least 50% identical, such        as at least 60%, such as at least 70%, at least 80%, at least        90%, at least 95% or at least 98% identical to SEQ ID NO: 11, or        a fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, such as at least 70%, at        least 80%, at least 90%, at least 95% or at least 98% identical        to SEQ ID NO: 12, or a fragment thereof having        beta-galactosidase activity; at least 50% identical, such as at        least 60%, such as at least 70%, at least 80%, at least 90%, at        least 95% or at least 98% identical to SEQ ID NO: 13, or a        fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, such as at least 70%, at        least 80%, at least 90%, at least 95% or at least 98% identical        to SEQ ID NO: 14, or a fragment thereof having        beta-galactosidase activity; or at least 50% identical, such as        at least 60%, such as at least 70%, at least 80%, at least 90%,        at least 95% or at least 98% identical to SEQ ID NO: 15, or a        fragment thereof having beta-galactosidase activity.    -   13. The formulation of any of the preceding embodiments, wherein        the polypeptide having beta-galactosidase activity has an amino        acid sequence which is at least 50%, such as at least 60%, at        least 70%, at least 80%, at least 90%, at least 95% or at least        98% identical to amino acids 1-1304 of SEQ ID NO: 1 or a        fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, at least 70%, at least 80%,        at least 90%, at least 95% or at least 98% identical to amino        acids 28-1931 of SEQ ID NO: 2 to amino acids 28-1931 of SEQ ID        NO: 2, or a fragment thereof having beta-galactosidase activity;        at least 50% identical, such as at least 60%, such as at least        70%, at least 80%, at least 90%, at least 95% or at least 98%        identical to amino acids 28-1331 of SEQ ID NO: 3, or a fragment        thereof having beta-galactosidase activity; at least 50%        identical, such as at least 60%, such as at least 70%, at least        80%, at least 90%, at least 95% or at least 98% identical to SEQ        ID NO: 4, or a fragment thereof having beta-galactosidase        activity; at least 50% identical, such as at least 60%, such as        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to SEQ ID NO: 5, or a fragment thereof        having beta-galactosidase activity; at least 50% identical, such        as at least 60%, such as at least 70%, at least 80%, at least        90%, at least 95% or at least 98% identical to SEQ ID NO: 13, or        a fragment thereof having beta-galactosidase activity; or at        least 50% identical, such as at least 60%, such as at least 70%,        at least 80%, at least 90%, at least 95% or at least 98%        identical to SEQ ID NO: 14, or a fragment thereof having        beta-galactosidase activity.    -   14. The formulation of any of the preceding embodiments, wherein        the polypeptide having beta-galactosidase activity has an amino        acid sequence which is at least 50%, such as at least 60%, at        least 70%, at least 80%, at least 90%, at least 95%, at least        98%, or 100% identical to amino acids 1-1304 of SEQ ID NO: 1 and        has a length of 900-1350 amino acids, preferably 1300-1305 amino        acids, more preferably 1302 or 1304 amino acids.    -   15. A polypeptide having beta-galactosidase activity having been        modified by glycation of at least one lysine and/or arginine        residue.    -   16. The polypeptide of embodiment 15, which has been modified by        glycation of at least 1%, preferably at least 3%, more        preferably at least 5%, even more preferably at least 10%, most        preferably at least 20%, of the lysine and arginine residues of        the polypeptide.    -   17. The polypeptide of any of embodiments 15-16 which has an        amino acid sequence which is at least 50%, such as at least 60%,        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to amino acids 1-1304 of SEQ ID NO: 1 or a        fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, at least 70%, at least 80%,        at least 90%, at least 95% or at least 98% identical to amino        acids 28-1931 of SEQ ID NO: 2, or a fragment thereof having        beta-galactosidase activity; at least 50% identical, such as at        least 60%, such as at least 70%, at least 80%, at least 90%, at        least 95% or at least 98% identical to amino acids 28-1331 of        SEQ ID NO: 3, or a fragment thereof having beta-galactosidase        activity; at least 50% identical, such as at least 60%, such as        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to SEQ ID NO: 4, or a fragment thereof        having beta-galactosidase activity; at least 50% identical, such        as at least 60%, such as at least 70%, at least 80%, at least        90%, at least 95% or at least 98% identical to SEQ ID NO: 5, or        a fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, such as at least 70%, at        least 80%, at least 90%, at least 95% or at least 98% identical        to SEQ ID NO: 6, or a fragment thereof having beta-galactosidase        activity; at least 50% identical, such as at least 60%, such as        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to SEQ ID NO: 7, or a fragment thereof        having beta-galactosidase activity; at least 50% identical, such        as at least 60%, such as at least 70%, at least 80%, at least        90%, at least 95% or at least 98% identical to SEQ ID NO: 8, or        a fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, such as at least 70%, at        least 80%, at least 90%, at least 95% or at least 98% identical        to SEQ ID NO: 9, or a fragment thereof having beta-galactosidase        activity; at least 50% identical, such as at least 60%, such as        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to SEQ ID NO: 10, or a fragment thereof        having beta-galactosidase activity; at least 50% identical, such        as at least 60%, such as at least 70%, at least 80%, at least        90%, at least 95% or at least 98% identical to SEQ ID NO: 11, or        a fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, such as at least 70%, at        least 80%, at least 90%, at least 95% or at least 98% identical        to SEQ ID NO: 12, or a fragment thereof having        beta-galactosidase activity; at least 50% identical, such as at        least 60%, such as at least 70%, at least 80%, at least 90%, at        least 95% or at least 98% identical to SEQ ID NO: 13, or a        fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, such as at least 70%, at        least 80%, at least 90%, at least 95% or at least 98% identical        to SEQ ID NO: 14, or a fragment thereof having        beta-galactosidase activity; or at least 50% identical, such as        at least 60%, such as at least 70%, at least 80%, at least 90%,        at least 95% or at least 98% identical to SEQ ID NO: 15, or a        fragment thereof having beta-galactosidase activity.    -   18. The polypeptide of any of embodiments 15-17 which has an        amino acid sequence which is at least 50%, such as at least 60%,        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to amino acids 1-1304 of SEQ ID NO: 1 or a        fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, at least 70%, at least 80%,        at least 90%, at least 95% or at least 98% identical to amino        acids 28-1931 of SEQ ID NO: 2 to amino acids 28-1931 of SEQ ID        NO: 2, or a fragment thereof having beta-galactosidase activity;        at least 50% identical, such as at least 60%, such as at least        70%, at least 80%, at least 90%, at least 95% or at least 98%        identical to amino acids 28-1331 of SEQ ID NO: 3, or a fragment        thereof having beta-galactosidase activity; at least 50%        identical, such as at least 60%, such as at least 70%, at least        80%, at least 90%, at least 95% or at least 98% identical to SEQ        ID NO: 4, or a fragment thereof having beta-galactosidase        activity; at least 50% identical, such as at least 60%, such as        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to SEQ ID NO: 5, or a fragment thereof        having beta-galactosidase activity; at least 50% identical, such        as at least 60%, such as at least 70%, at least 80%, at least        90%, at least 95% or at least 98% identical to SEQ ID NO: 13, or        a fragment thereof having beta-galactosidase activity; or at        least 50% identical, such as at least 60%, such as at least 70%,        at least 80%, at least 90%, at least 95% or at least 98%        identical to SEQ ID NO: 14, or a fragment thereof having        beta-galactosidase activity.    -   19. The polypeptide of any of embodiments 15-18 which has an        amino acid sequence which is at least 50%, such as at least 60%,        at least 70%, at least 80%, at least 90%, at least 95%, at least        98%, or 100% identical to amino acids 1-1304 of SEQ ID NO: 1 and        has a length of 900-1350 amino acids, preferably 1300-1305 amino        acids, more preferably 1302 or 1304 amino acids.    -   20. A method of modifying a polypeptide having        beta-galactosidase activity comprising contacting the        polypeptide with a reducing sugar, preferably fructose, glucose,        galactose, or lactose for a time and temperature sufficient to        produce a polypeptide modified by glycation.    -   21. The method of embodiment 20 which is a method of modifying        by glycation a polypeptide having beta-galactosidase activity.    -   22. The method of any of embodiments 20-21, wherein the        polypeptide having beta-galactosidase activity modified by        glycation has improved transgalactosylating activity as compared        to the polypeptide having beta-galactosidase activity which has        not been modified by glycation.    -   23. The method of any of embodiments 20-22, wherein the        polypeptide having beta-galactosidase activity is modified by        glycation of at least 1%, preferably at least 3%, more        preferably at least 5%, even more preferably at least 10%, most        preferably at least 20%, of the lysine and arginine residues of        the polypeptide.    -   24. The method of any of embodiments 20-23, comprising        contacting the polypeptide having beta-galactosidase activity        with 30-90 wt % of a reducing sugar, preferably fructose,        glucose, or galactose, at pH 5-8 for a time of 3-100 hours at a        temperature of 20-80° C.    -   25. The method of any of embodiments 20-24, comprising        contacting the polypeptide having beta-galactosidase activity at        pH 5-8, preferably pH 6-7, for a time of 3-100 hours, preferably        15-80 hours, at a temperature of 50-80° C., preferably 50-70° C.    -   26. The method of any of embodiments 20-25, comprising        contacting the polypeptide having beta-galactosidase activity        with 30-90 wt %, preferably 40 wt %, 60 wt %, or 80 wt % of a        reducing sugar, preferably glucose.    -   27. The method of any of embodiments 20-25, comprising        contacting the polypeptide having beta-galactosidase activity        with 30-90 wt %, preferably 40-65 wt %, of a reducing sugar.    -   28. The method of any of embodiments 20-27, wherein the reducing        sugar is fructose, glucose or galactose, preferably glucose.    -   29. The method of any of embodiments 20-28, wherein the        polypeptide having beta-galactosidase activity has an amino acid        sequence which is at least 50%, such as at least 60%, at least        70%, at least 80%, at least 90%, at least 95% or at least 98%        identical to amino acids 1-1304 of SEQ ID NO: 1 or a fragment        thereof having beta-galactosidase activity; at least 50%        identical, such as at least 60%, at least 70%, at least 80%, at        least 90%, at least 95% or at least 98% identical to amino acids        28-1931 of SEQ ID NO: 2, or a fragment thereof having        beta-galactosidase activity; at least 50% identical, such as at        least 60%, such as at least 70%, at least 80%, at least 90%, at        least 95% or at least 98% identical to amino acids 28-1331 of        SEQ ID NO: 3, or a fragment thereof having beta-galactosidase        activity; at least 50% identical, such as at least 60%, such as        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to SEQ ID NO: 4, or a fragment thereof        having beta-galactosidase activity; at least 50% identical, such        as at least 60%, such as at least 70%, at least 80%, at least        90%, at least 95% or at least 98% identical to SEQ ID NO: 5, or        a fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, such as at least 70%, at        least 80%, at least 90%, at least 95% or at least 98% identical        to SEQ ID NO: 6, or a fragment thereof having beta-galactosidase        activity; at least 50% identical, such as at least 60%, such as        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to SEQ ID NO: 7, or a fragment thereof        having beta-galactosidase activity; at least 50% identical, such        as at least 60%, such as at least 70%, at least 80%, at least        90%, at least 95% or at least 98% identical to SEQ ID NO: 8, or        a fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, such as at least 70%, at        least 80%, at least 90%, at least 95% or at least 98% identical        to SEQ ID NO: 9, or a fragment thereof having beta-galactosidase        activity; at least 50% identical, such as at least 60%, such as        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to SEQ ID NO: 10, or a fragment thereof        having beta-galactosidase activity; at least 50% identical, such        as at least 60%, such as at least 70%, at least 80%, at least        90%, at least 95% or at least 98% identical to SEQ ID NO: 11, or        a fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, such as at least 70%, at        least 80%, at least 90%, at least 95% or at least 98% identical        to SEQ ID NO: 12, or a fragment thereof having        beta-galactosidase activity; at least 50% identical, such as at        least 60%, such as at least 70%, at least 80%, at least 90%, at        least 95% or at least 98% identical to SEQ ID NO: 13, or a        fragment thereof having beta-galactosidase activity; at least        50% identical, such as at least 60%, such as at least 70%, at        least 80%, at least 90%, at least 95% or at least 98% identical        to SEQ ID NO: 14, or a fragment thereof having        beta-galactosidase activity; or at least 50% identical, such as        at least 60%, such as at least 70%, at least 80%, at least 90%,        at least 95% or at least 98% identical to SEQ ID NO: 15, or a        fragment thereof having beta-galactosidase activity.    -   30. The method of any of embodiments 20-29, wherein the        polypeptide having beta-galactosidase activity has an amino acid        sequence which is at least 50%, such as at least 60%, at least        70%, at least 80%, at least 90%, at least 95% or at least 98%        identical to amino acids 1-1304 of SEQ ID NO: 1 or a fragment        thereof having beta-galactosidase activity; at least 50%        identical, such as at least 60%, at least 70%, at least 80%, at        least 90%, at least 95% or at least 98% identical to amino acids        28-1931 of SEQ ID NO: 2 to amino acids 28-1931 of SEQ ID NO: 2,        or a fragment thereof having beta-galactosidase activity; at        least 50% identical, such as at least 60%, such as at least 70%,        at least 80%, at least 90%, at least 95% or at least 98%        identical to amino acids 28-1331 of SEQ ID NO: 3, or a fragment        thereof having beta-galactosidase activity; at least 50%        identical, such as at least 60%, such as at least 70%, at least        80%, at least 90%, at least 95% or at least 98% identical to SEQ        ID NO: 4, or a fragment thereof having beta-galactosidase        activity; at least 50% identical, such as at least 60%, such as        at least 70%, at least 80%, at least 90%, at least 95% or at        least 98% identical to SEQ ID NO: 5, or a fragment thereof        having beta-galactosidase activity; at least 50% identical, such        as at least 60%, such as at least 70%, at least 80%, at least        90%, at least 95% or at least 98% identical to SEQ ID NO: 13, or        a fragment thereof having beta-galactosidase activity; or at        least 50% identical, such as at least 60%, such as at least 70%,        at least 80%, at least 90%, at least 95% or at least 98%        identical to SEQ ID NO: 14, or a fragment thereof having        beta-galactosidase activity.    -   31. The method of any of embodiments 20-30, wherein the        polypeptide having beta-galactosidase activity has an amino acid        sequence which is at least 50%, such as at least 60%, at least        70%, at least 80%, at least 90%, at least 95%, at least 98%, or        100% identical to amino acids 1-1304 of SEQ ID NO: 1 and has a        length of 900-1350 amino acids, preferably 1300-1305 amino        acids, more preferably 1302 or 1304 amino acids.    -   32. A method for producing galacto-oligosaccharides (GOS)        comprising contacting the formulation of any of embodiments 1-14        or the polypeptide of any of embodiments 15-19 or a polypeptide        having beta-galactosidase activity which has been modified by        the method of any of claims 20-31 with lactose.    -   33. A method for producing galacto-oligosaccharides (GOS)        comprising contacting a polypeptide having a sequence comprising        or consisting of amino acids 1-1304 of SEQ ID NO: 1, with        lactose under conditions of high temperature and high initial        lactose concentration.    -   34. The method of embodiment 33, wherein the temperature is        40-80° C., such as 50° C., 60° C., 65° C., 70° C., 75° C., or        80° C. and wherein the initial lactose concentration is above        40% (w/w), such as 40-50% (w/w), 45% (w/w), 50% (w/w), 55%        (w/w), 40-60% (w/w) or even above 60% (w/w), such as 61% (w/w),        62% (w/w), 63% (w/w), 64% (w/w), 65% (w/w), 66% (w/w), 67%        (w/w), 68% (w/w), 69% (w/w), 70% (w/w), 71% (w/w), 72% (w/w),        73% (w/w), 74% (w/w), 75% (w/w), or 80% (w/w) lactose.

EXAMPLES Materials and Methods Activity Assay (LAU(C)) Principle:

Lactase hydrolyzes lactose to form α-D-glucose. The α-D-glucose isphosphorylated by ATP, in a reaction catalyzed by hexokinase. Theglucose-6-phosphate formed is oxidized to 6-phosphogluconate byglucose-6-phosphate dehydrogenase. Concomitant with this reaction anequimolar amount of NAD+ is reduced to NADH with a resulting increase inabsorbance at 340 nm.

Reagents:

15% (w/v) Brij L23: Weigh out 508.0±0.4 g of Brij® L23 (Sigma B4184)into a beaker. Add approx. 300 mL ultrapure water and stir. Transfer theBrij® L23 quantitatively to a 1 L volumetric flask. Fill to the markwith ultrapure water. Stir until homogenous. Storability: 2 months inrefrigerator.

Colour reagent: (Glucose reagent kit (GHK) (0.1 M Tris, 2.1 mM ATP, 2.1mM NAD, 4 mM Mg2+, <0.1% NaN3, 4 mMMg2+, >7.5 kU/L hexokinase, >7.5 kU/LG-6-P-DH, pH 7.8)): Open a vial of Glucose (HK) Reagent A, Thermo FisherScientific (Art. no.: 981304 or 981779) and a vial of Glucose (HK)Reagent B, Thermo Fisher Scientific (Art. no.: 981304 or 981779). Pour 1vial of reagent B into 1 vial of reagent A. Put on the cap. Mix well byslowly and gently turning up and down the vial 10-15 times. Use thewhole mixture in reagent A vial, or pour needed amount into anappropriate container. Storability: 1 month in refrigerator. Dissolutionbuffer/dilution buffer (0.01 M Citric acid monohydrate, 0.0225% (w/v)Brij® L23, 1 mM MgSO4, 7H2O, pH 4.5): Weigh out 21.0±0.1 g of Citricacid monohydrate (Cas. No. 5949-29-1) and transfer quantitative to a 10L volumetric flask. Weigh out 2.46±0.01 g of MgSO4, 7H2O (Cas. no.10034-99-8) and transfer quantitative to the volumetric flask. Addapproximately 9 L of demineralized water and stir until completelydissolved. Add 15 mL of 15% (w/v) Brij L23 to the volumetric flask andstir. Add approximately 35 mL of 4 M NaOH (Cas. No. 1310-73-2) and stir.Adjust pH to 4.50±0.05 using e.g. 4 M NaOH or e.g. HCl as appropriate.Fill to the mark with demineralized water and stir. Storability: 13 daysat room temperature.

Substrate (31.6% w/w lactose monohydrate, 0.01M citric monohydrate,0.0225 (w/v) Brij L23, 1 mM MgSO4, 7H2O): Weigh out 7.9±0.2 g of Lactosemonohydrate (Cas. No. 10039-26-6) directly into a beaker. Dissolve to atotal volume of 25.0±0.1 g of dissolution buffer. Heat up and stir untilfully dissolved with no boiling of the substrate. Storability: 6 hoursat room temperature.

Standard: Enzyme standard with identified LAU(C)/g (available fromNovozymes NS, Denmark) is used as standard, diluted in dissolutionbuffer in the range from 0.197-0.7880 LAU(C)/mL.

Procedure:

1. 50 uL of substrate is incubated for 540 seconds at 50° C. Blank (50uL of dissolution buffer) is subtracted out.

2. 25 uL sample in dissolution buffer is added.

3. The reaction is incubated for 1800 seconds followed by addition of160 uL colour reagent.

4. After 300 seconds, the absorbance is measured at 340 nm.

Calculation of Enzyme Activity: The enzyme activity of the dilutedsample is read from the standard curve. Calculation of activity of asample in LAU(C)/g is as stated in the formula:

Activity Unit/g=S·V·F/W

S=Reading from the standard curve in LAU(C)/mL

V=Volume of the measuring flask used in mL

F=Dilution factor for second dilution

W=Weight of sample in g

Application in Yoghurt

Commercial homogenized milk with 1.5% fat is pasteurized at 90° C. for20 min. 200 ml of the milk is transferred into baby bottles and temperedto 43° C. The milk is inoculated with a frozen probiotic yoghurt culturee.g. Chr. Hansen, Denmark, (F-DVS ABY-3) using an inoculation level of0.02%. At the same time, enzyme is added to the milk. The milk samplesare fermented at 43° C. until pH reached 4.55 within approximately fivehours. The yoghurts are then stirred, cooled to 25° C. and placed at 8°C. for storage. Samples are collected 2 hours after addition of cultureand enzyme, at end pH (pH 4.55) and after 20-24 hours (Day 1) of storageat 8° C. The biological activity is stopped by addition of sulphuricacid. Proteins are precipitated adding perchloric acid and MQWcontaining standards are then added. Lactose hydrolysis is measuredusing a Dionex ICS-3000 system equipped with a Carbopac20 connected withan electrochemical detector (ED). Peaks are identified and quantified bycomparing with known standards of lactose, glucose and galactose.Content of DP2 saccharides, particularly lactose, and GOS in the form ofDP3+ are identified and quantified. Vivinal GOS (Friesland Campina) is auseful standard for GOS quantification.

Application in Yoghurt

Commercial homogenized milk with 1.5% fat is pasteurized at 90° C. for20 min. 200 ml of the milk is transferred into baby bottles and temperedto 43° C. The milk is inoculated with a frozen probiotic yoghurt culturee.g. Chr. Hansen, Denmark, (F-DVS ABY-3) using an inoculation level if0.02%. At the same time enzyme is added to the milk. The milk samplesare fermented at 43° C. until pH reached 4.55 within approximately fivehours. The yoghurts are then stirred, cooled to 25° C. and placed at 8°C. for storage. Samples are collected 2 hours after addition of cultureand enzyme, at end pH (pH 4.55) and after 1, 2, 3 and 7 days of storageat 8° C. The biological activity is stopped by addition of sulphuricacid. Proteins are precipitated adding perchloric acid and MQWcontaining standards are then added. Lactose hydrolysis is measuredusing a Dionex ICS-3000 system equipped with a Carbopac20 connected withan electrochemical detector (ED). Peaks are identified and quantified bycomparing with known standards of lactose, glucose and galactose.Content of DP2 saccharides, particularly lactose, and GOS in the form ofDP3+ are identified and quantified. Vivinal GOS (Friesland Campina) is auseful standard for GOS quantification.

Application in 1.5% Milk

Commercial homogenized milk with 1.5% fat is transferred to tubes (10ml) and heated in water baths to 40° C., 50° C. and 55° C.,respectively. Enzyme is then added to the milk samples. Samples arecollected 2 hours and 4 hours after addition of the enzyme. Thebiological activity is stopped by addition of sulphuric acid. Proteinsare precipitated adding perchloric acid and MQW containing standards isthen added. Lactose hydrolysis is measured using a Dionex ICS-3000system equipped with a Carbopac20 connected with an electrochemicaldetector (ED). Peaks are identified and quantified by comparing withknown standards of lactose, glucose and galactose. Content of DP2saccharides, particularly lactose, and GOS in the form of DP3+ areidentified and quantified. Vivinal GOS (Friesland Campina) is a usefulstandard for GOS quantification.

Application in Skimmed Milk Solution

100 ml 9% skimmed milk solution having approximately 5% lactose is madeby mixing 9 g skimmed milk powder (Kerry) in 91 ml ionic water. 10 ml ofthe solution is transferred to a test tube containing a magneticstirring bar and placed in a water bath at 37° C. After 15 min enzyme isadded. Milk samples are taken at regular intervals up till 4 hrs. andthe enzyme inactivated by heating to 99° C. for 10 min in a thermomixer.Samples are diluted appropriately and filtered through a 0.20 um filter.Lactose hydrolysis is measured using a Dionex BioLC equipped with aDionex PA1 column and a Pulsed Amperiometrisk Detektor (PAD). Peaks areidentified and quantified by comparing with known standards of lactose,glucose and galactose. Content of DP2 saccharides, particularly lactose,and GOS in the form of DP3+ are identified and quantified. Vivinal GOS(Friesland Campina) is a useful standard for GOS quantification.

Application in 1.5% Milk—High Temperature

Commercial homogenized milk with 1.5% fat is transferred to tubes (10ml) and tempered to 63° C. Enzyme is added to the milk samples. At 63°C. samples are collected 15 minutes, 30 minutes, 2 hours and 4 hoursafter addition of the enzyme. The enzymatic activity in the samples isstopped by addition of sulphuric acid and proteins precipitated byaddition of perchloric acid before HPLC analysis. Lactose hydrolysis ismeasured using a Dionex ICS-3000 system equipped with a Carbopac20connected with an electrochemical detector (ED). Peaks are identifiedand quantified by comparing with known standards of lactose, glucose andgalactose. Content of DP2 saccharides, particularly lactose, and GOS inthe form of DP3+ are identified and quantified. Vivinal GOS (FrieslandCampina) is a useful standard for GOS quantification.

Application in Whey Permeate Solution

100 ml 15 or 30% (w/w) whey permeate containing primarily lactose andions is made by mixing 15 or 30 g spray-dried whey permeate powder(Variolac, Arla) in 85 or 70 ml ionic water respectively. The solutionis poured in a flask containing a magnetic stirring bar and placed in awater bath at 37° C. After 15 min, enzyme is added. Milk samples aretaken at regular intervals up till 5.5 hrs. and the enzyme inactivatedby heating to 99° C. for 10 min in a thermomixer. Samples are dilutedappropriately and filtered through a 0.20 um filter. Lactose hydrolysisis measured using a Dionex BioLC equipped with a Dionex PA1 column and aPulsed Amperiometrisk Detektor (PAD). Peaks are identified andquantified by comparing with known standards of lactose, glucose andgalactose. Content of DP2 saccharides, particularly lactose, and GOS inthe form of DP3+ are identified and quantified. Vivinal GOS (FrieslandCampina) is a useful standard for GOS quantification.

Example 1

Production of Polypeptide

Bifidobacterium bifidum β-galactosidase (BBB) having the sequence shownas SEQ ID NO: 1 is expressed in Bacillus licheniformis

Example 2

Glycation

BBB-un_1: Untreated Bifidobacterium bifidum β-galactosidase (BBB-un_1)is expressed in Bacillus licheniformis according to Example 1 andconcentrated using ultra filtration (cut-off 10 kDa) and finallyformulated with glycerol 50% (w/w). Activity of this sample is 7210LAU(C)/g.

To a 100 ml Distec vessel, 100 grams of 66% (w/w) sugar (glucose (Glc),galactose (Gal) or lactose (Lac)) solution with 20 mM succinic acidbuffer pH 6.5 is added and preheated to 60° C. for 15 min. Then 10 mlBBB-un_1 without glycerol is added and incubated at 60° C. with mixingfor 16 hr. The solution is cooled to room temperature and dialyzed(cut-off 12 kDa) against 5 mM succinic acid buffer pH 6.5 for 16 hr at5° C. and then concentrated to ˜5 ml using Amicon cell cut-off 10 kDaand finally added the same volume of glycerol to give a conc. of 50%glycerol (v/v). The three samples generated from this procedure aretermed BBB-Glc, BBB-Gal and BBB-Lac referring to the sugar used for theincubation.

Filter-aided sample preparation (FASP) MS data from tryptic digests wasmade and glycated peptides were identified as Lys and Arg+1 Hexose,causing 1 missed cleavage site. The % of glycated trypsin digestedpeptides were estimated to be 0.66%, 31%, 36% and 27% for untreated,lactose treated, glucose treated and galactose treated respectively.Thus, mass spectrometery of peptides made from trypsin digest confirmsglycation on lysine and arginine residues of BBB-Glc, BBB-Gal andBBB-Lac but these glycations are not present in BBB-un 1.

GOS Production at 25° C.

To evaluate GOS produced at 25° C., 50 ul 1280 LAU(C)/g enzyme(BBB-un_1, BBB-Glc, BBB-Gal or BBB-Lac) is mixed with 950 ul preheated66.5% lactose*H2O (w/w), 20 mM succinate pH 6.5 in an Eppendorf tubewhich gives a final concentration of 60% lactose. This mixture is thenincubated at 25° C. with 1000 rpm for 22 hr and applied on ice.Inactivation of the enzyme is then performed by diluting the 1 ml GOSproduct with 49 ml 0.04 M NaOH, 1 mM EDTA and incubated for 5 min atroom temp. Then an additional 40× dilution with milli Q water (i.e.2000× dilution in total) is made and applied to a PA1 column(High-Performance Anion-Exchange Chromatography) with PulsedAmperometric Detection (HPAEC-PAD).

GOS Production at 65° C.

In order to evaluate GOS produced at 65° C., 50 ul 192 LAU(C)/g enzyme(BBB-un_1, BBB-Glc, BBB-Gal or BBB-Lac) is mixed with 950 ul preheated66.5% lactose*H20 (w/w), 20 mM succinate pH 6.5 in an Eppendorf tubewhich gives a final concentration of 60% lactose. This mixture is thenincubated at 65° C. with 1000 rpm for 22 hr and applied on ice.Inactivation of the enzyme is then performed by diluting the 1 ml GOSproduct with 49 ml 0.04 M NaOH, 1 mM EDTA and incubated for 5 min atroom temp. Then an additional 40× dilution with milli Q water (i.e.2000× dilution in total) is made and applied to a PA1 column(High-Performance Anion-Exchange Chromatography) with PulsedAmperometric Detection (HPAEC-PAD).

TABLE 1 (Glc-Gal)/Gal (Glc-Gal)/Gal 25° C. 65° C. BBB-un_1 0.79 10BBB-Lac 6.9 12 BBB-Glc 7.8 13 BBB-Gal 6.0 12

As seen in Table 1, untreated Bifidobacterium bifidum beta-galactosidase(BBB-un_1) has low transgalactosylating activity at 25° C. with a(Glc-Gal)/Gal ratio of 0.79 compared with the glycated BBB forms(BBB-Glc, BBB-Gal and BBB-Lac) which have 7-10 fold higher (Glc-Gal)/Galratio when incubated at the same process conditions. At 65° C., thedifference is less pronounced between untreated and glycated BBB andonly a 1.2-1.3 fold increase in (Glc-Gal)/Gal ratio is seen. However, itis surprising that all enzyme BBB-un_1, BBB-Glc, BBB-Gal and BBB-Lachave a pronounced increase in (Glc-Gal)/Gal ratio at elevatedtemperature, 65° C. compared to 25° C., especially for BBB-un which hasa 13-fold increase in (Glc-Gal)/Gal ratio.

Example 3

Sample:

BBB-un_2: Untreated Bifidobacterium bifidum β-galactosidase (BBB-un_2)is expressed in Bacillus licheniformis according to Example 1 andconcentrated using ultrafiltration (cut-off 10 kDa) and finallyformulated with glucose 40% (w/w), 60% (w/w) or 80% (w/w), with anenzyme concentration of 7575 LAU(C)/g, 9200 LAU(C)/g and 4600 LAU(C)/g,respectively.

Glycation of Enzyme Samples:

Enzyme solution formulated with glucose are incubated for 16 h and 40 hat three different temperatures 50° C., 55° C. and 60° C., see Table 2.

GOS Production at 25° C.

To evaluate GOS produced at 25° C., 50 μl enzyme sample as shown inTable 2 is mixed with 950 μl preheated 66.5% lactose*H20 (w/w), 20 mMsuccinate pH 6.5 in an Eppendorf tube which gives a final concentrationof 60% lactose. This mixture is then incubated at 25° C. with 1000 rpmfor 22 hr and applied on ice. Inactivation of the enzyme is thenperformed by diluting the 1 ml GOS product with 49 ml 0.04 M NaOH, 1 mMEDTA and incubated for 5 min at room temp. Then an additional 40×dilution with milli Q water (i.e. 2000× dilution in total) is made andapplied to a PA1 column (which is High-Performance Anion-ExchangeChromatography, HPAEC) and carbohydrates were detected with PulsedAmperometric Detection (PAD).

Results & Discussion

TABLE 2 Glucose Temperature Time conc. (Glc-Gal)/Gal LAU(C)/g ° C. h %Ratio 7575 No heat 40 0.5 treatment 7575 50 16 40 0.9 7575 55 16 40 0.97575 60 16 40 1.4 7575 50 40 40 1.6 7575 55 40 40 2.1 7575 60 40 40 2.59200 No heat 60 0.5 treatment 9200 50 16 60 0.9 9200 55 16 60 1.4 920060 16 60 2.0 9200 50 40 60 2.2 9200 55 40 60 2.9 9200 60 40 60 3.3 460050 16 80 1.9 4600 55 16 80 4.3 4600 60 16 80 5.0 4600 50 40 80 5.3 460055 40 80 6.1 4600 60 40 80 6.1

Table 2 shows that there is an increase in the (Glc-Gal)/Gal ratio whenthe temperature is increased from 50 to 60° C. and the (Glc-Gal)/Galratios are higher than control where no heat treatment has been made.Prolonged incubation times also increases the (Glc-Gal)/Gal ratio, i.e.higher values are obtained after 40 h compared to 16 h. The effect ofthe temperature and time is the same for enzymes formulated with 40%,60% and 80% glucose.

Example 4

Glycation of Enzyme Samples

Untreated Bifidobacterium bifidum β-galactosidase (BBB-un) is expressedin Bacillus licheniformis according to Example 1 and concentrated usingultrafiltration (cut-off 10 kDa) and formulated with glucose at levelsas indicated in the table by incubating for 44 h at 55° C. then storedat 4° C.

GOS Production in Milk at 5° C.

One ml semi-skim milk is applied in 2 ml Eppendorf tube and heated to90° C. for 5 min and cooled in ice-bath for at least 30 min. Then 10 μldiluted enzyme sample is added and incubated for 24 h at 5° C. Thereaction is stopped by adding 5 μl HAc and heated to 90° C. for 5 minand centrifuged at 20,000 g for 5 min. Then 50 μl supernatant is addedto 500 μl Milli Q water+10 μl Carrez I solution in a 5 ml Eppendorf tubeand mixed followed by adding 10 μl Carrez II solution and mixed. Then4.43 ml milli Q water is added and centrifuged at 20,000 g for 5 min atroom temperature. Then 1 ml of supernatant is added to 4 ml water andfiltered through a 0.20 μm filter into a HPLC vial and applied to a PA1HPAEC column and carbohydrates are detected with PAD.

GOS Production in Milk at 42° C.

One ml semi-skim milk is applied in 2 ml Eppendorf tube and heated to90° C. for 5 min and cooled in ice-bath for at least 30 min. Then 10 μldiluted enzyme sample is added and incubated for 6 h at 42° C. Thereaction is stopped by adding 5 μl HAc and heated to 90° C. for 5 minand centrifuged at 20,000 g for 5 min. Then 50 μl supernatant is addedto 500 μl Milli Q water+10 μl Carrez I solution in a 5 ml Eppendorf tubeand mixed followed by adding 10 μl Carrez II solution and mixed. Then4.43 ml milli Q water is added and centrifuged at 20,000 g for 5 min atroom temperature. Then 1 ml of supernatant is added to 4 ml water andfiltered through a 0.20 μm filter into a HPLC vial and applied to a PA1HPAEC column and carbohydrates are detected with PAD.

GOS Production in 35% Reconstituted Skim Milk Powder at 42° C.

One ml 35% (w/w) reconstituted skim milk powder is applied in 2 mleppendorf tube and heated to 90° C. for 5 min and cooled in ice-bath forat least 30 min. Then 10 μl diluted enzyme sample is added and incubatedfor 6 h at 42° C. The reaction is stopped by adding 5 μl HAc and heatedto 90° C. for 5 min and centrifuged at 20,000 g for 5 min. Then 50 μlsupernatant is added to 500 μl Milli Q water+10 μl Carrez I solution ina 5 ml Eppendorf tube and mixed followed by adding 10 μl Carrez IIsolution and mixed. Then 4.43 ml milli Q water is added and centrifugedat 20,000 g for 5 min at room temperature. Then 0.35 ml of supernatantis added to 4.65 ml water and filtered through a 0.20 μm filter into aHPLC vial and applied to a PA1 column (which is High-PerformanceAnion-Exchange Chromatography, HPAEC) and carbohydrates are detectedwith Pulsed Amperometric Detection (PAD).

Results & Discussion

TABLE 3 Data from GOS production in milk at 5° C. Glucose (Glc-Gal)/Enzyme amount Temperature Time conc. Gal mean LAU(C)/mL ° C. h % ratiodev. 640 55 44 40 0.34 0.01 640 55 44 60 1.06 0.03 640 No heat treatment40 0.07 0.002

TABLE 4 Data from GOS production in milk at 42° C. Enzyme Glucose(Glc-Gal)/ amount Temperature Time conc. Gal mean LAU(C)/mL ° C. h %ratio dev. 290 55 44 40 0.31 0.02 290 55 44 60 0.51 0.02 290 No heattreatment 40 0.05 0.004

TABLE 5 Data from GOS production in 35% (w/w) reconstituted skim milkpowder at 42° C. Enzyme Glucose (Glc-Gal)/ amount Temperature Time conc.Gal mean LAU(C)/mL ° C. h % ratio dev. 840 55 44 40 0.68 0.005 840 55 4460 1.8 0.2 840 No heat treatment 40 0.07 0.003

Table 3, 4 and 5 shows that (Glc-Gal)/Gal ratio is increased for both40% and 60% glucose formulations when incubated at 55° C. for 44 hcompared to 40% glucose control (No heat treatment). These results showthat GOS can be generated in-situ (in milk) at 5° C. which is the commonstorage temperature of milk but also at 42° C. which is useful foryoghurt application as 42° C. is a common fermentation temperature. Aneven higher (Glc-Gal)/Gal ratio can be achieved in 35% (w/w)reconstituted skim milk powder (table 5), i.e. increasing the dry mattercontent and lactose concentration therefore increasing transferaseefficiency.

Example 5

Glycosylation of Enzyme Samples

Untreated Bifidobacterium bifidum β-galactosidase (BBB) having thesequence shown as SEQ ID NO: 1 is expressed in Bacillus licheniformisand concentrated using ultrafiltration (cut-off 10 kDa) to 23000LAU(B)/g and formulated with either 60% (w/w) glucose (3 gram glucose+2gram BBB-un) or 60% (w/w) glycerol (3 gram glycerol+2 gram BBB-un) andincubated for 66 h at 50° C. or unformulated diluted with water withsame dilution i.e. 3 gram water+2 gram BBB-un and incubated for 30 minat 50° C.

GOS Production at 25° C.

To evaluate GOS produced at 25° C., 50 ul 770 LAU(B)/g enzyme is mixedwith 950 ul preheated 66.5% lactose*H20 (w/w), 20 mM succinate pH 6.5 inan Eppendorf tube which gives a final concentration of 60% lactose. Thismixture is then incubated at 25° C. with 1000 rpm for 22 hr and appliedon ice. Inactivation of the enzyme is then performed by diluting the 1ml GOS product with 9 ml 0.04 M NaOH and incubated for 5 min at roomtemp. Then an additional 200× dilution with milli Q water (i.e. 2000×dilution in total) is made and applied to a PA1 column (High-PerformanceAnion-Exchange Chromatography) with Pulsed Amperometric Detection(HPAEC-PAD).

TABLE 6 Enzyme (Glc-Gal)/ amount Temperature Time Gal mean FormulationLAU(B)/g ° C. h ratio dev. BBB treated in 770 50 66 5.5 0.1 60% glucoseBBB treated in 770 50 66 0.70 0.05 60% glycerol BBB treated in 770 500.5 0.68 0.01 water

The incubation in 60% glucose is made at 50° C. for 66 h to ensureglycation of the Bifidobacterium bifidum β-galactosidase (BBB).Incubation in 60% glycerol (which is not a reducing sugar) is includedas a control. The sample without formulating agent (BBB treated inwater) is included as another control. Due to instability of the enzymewhen no stabilizer is added (e.g. glucose or glycerol), the enzyme wouldnot be stable for 66 h at 50° C. and therefore the “BBB treated inwater” sample was incubated only for 0.5 h at 50° C.

Table 6 shows that only high (Glc-Gal)/Gal ratio (5.5) is obtained byincubating Bifidobacterium bifidum β-galactosidase (BBB) with glucoseand not with controls formulated in glycerol or without formulationagents (water). Thus, these results show that it is not heating of theenzyme sample as such that transforms the enzyme to get a high(Glc-Gal)/Gal ratio. It is the incubation with glucose at conditionsthat enable glycation of the enzyme that ensures the transformation fromlow to high (Glc-Gal)/Gal ratio.

Example 6

Sample:

BBB-1: Bifidobacterium bifidum β-galactosidase having the sequence shownas SEQ ID NO: 1 has been expressed in Bacillus licheniformis and columnpurified and then finally formulated with 60% glucose (BBB-1-G) andincubated for 66 h at 50° C. as shown in Table 6 and then stored at −20°C. Control sample (BBB-1-C) were not formulated with glucose and juststored at −20° C.

Kluyveromyces lactis β-galactosidase (Lactozym® Pure) has been expressedin Kluyveromyces lactis and concentrated using UF (cut-off 10 kDa) andfinally formulated with 60% glucose with same enzyme protein conc.([ep]) as BBB-1-G and incubated for 66 h at 50° C. as shown in Table 6and then stored at −20° C. Control sample was not formulated withglucose and just stored at −20° C. and has the same enzyme protein conc.([ep]) as BBB-1-C.

Bacillus circulans β-galactosidase having the sequence shown as aminoacids 28-1737 of SEQ ID NO: 14 has been expressed in Bacillus subtilisand column purified and then finally formulated with 60% glucose withsame enzyme protein conc. ([ep]) as BBB-1-G and incubated for 66 h at50° C. as shown in Table 6 and then stored at −20° C. Control sample wasnot formulated with glucose and just stored at −20° C. and has the sameenzyme protein conc. ([ep]) as BBB-1-C.

BBB-2: Bifidobacterium bifidum β-galactosidase having the sequence shownas SEQ ID NO: 1 has been expressed in Bacillus licheniformis andconcentrated using UF (cut-off 10 kDa) and finally formulated with 50%glycerol and incubated for 4 weeks (672 h) at 40° C. as shown in Table 6and then stored at −20° C. Control sample was not incubated at 40° C.but just stored at −20° C. during the 4 weeks

BBB-3: Bifidobacterium bifidum β-galactosidase having the sequence shownas SEQ ID NO: 1 has been expressed in Bacillus licheniformis andconcentrated using UF (cut-off 10 kDa) and finally formulated with 40%glucose and incubated for 4 weeks (672 h) at 40° C. as shown in Table 6and then stored at −20° C. Control sample was not incubated at 40° C.but just stored at −20° C. during the 4 weeks

GOS Production in Regular Milk

5° C. for 24 h (Results are Shown in Table 6).

Two ml semi-skim milk (Arla, purchased in a local Danish supermarket,4.7 g lactose and 3.5 g protein per 100 g) was transferred into a 5 mlEppendorf tube (double determinations for each dose including control).Then 20 μl of enzyme dilution was added (see Table 6) and mixed followedby an incubation at 5° C. for 24 h. After incubation, 10 μl concentratedacetic acid was added to each sample and the solutions were heated to90° C. for 5 min. After inactivation, the samples were centrifuged at14,000 rpm for 5 min at room temperature. One ml supernatant wastransferred to another tube and kept frozen until analysed by HPLC.

Determination of Ratio of (Glc-Gal)/Gal

High-Performance Anion-Exchange Chromatography with Pulsed AmperometricDetection (HPAEC-PAD) using a PA1 column for quantitative determinationof galactose (Gal) and glucose (Glc) is performed as follows.

50 μl sample is mixed together with 500 μl Milli Q water+10 μl Carrez Isolution in a 5 ml Eppendorf tube, and then mixed with 10 μl Carrez IIsolution. Then 4.43 ml milli Q water is added and centrifugated at14,000 rpm for 5 min at room temperature. One ml supernatant is mixedwith 4 ml Milli-Q water and filtered through a 0.2 μm filter into a HPLCvial and applied on a PA1 column. Quantitative determination of Glc andGal was made using a known standard of Glc and Gal, respectively.

TABLE 6 Enzyme Temper- (Glc-Gal)/ amount ature Time Gal mean LAU(B)/g °C. h ratio dev. BBB-1-G 260 50 66 3.39 0.03 (60% glucose) K. lactis“same [ep] 50 66 0.40 0.02 (60% glucose) as BBB-1-G” B. circulans “same[ep] 50 66 4.09 0.07 (60% glucose) as BBB-1-G” BBB-1-C- 640 No heattreatment −0.04 0.005 control K. lactis - “same [ep] No heat treatment0.00 0.02 control as BBB-1-C” B. circulans - “same [ep] No heattreatment 0.00 0.001 control as BBB-1-C” BBB-2 640 40 672 −0.02 0.02(50% glycerol) BBB-3 640 40 672 0.75 0.14 (40% glucose) BBB-2 640 −20672 −0.02 0.02 (50% glycerol) - control BBB-3 640 −20 672 −0.01 0.01(40% glucose) - control

A pronounced increase in (Glc-Gal)/Gal ratio is seen whenBifidobacterium bifidum β-galactosidase and Bacillus circulansβ-galactosidase (both GH2_5) is incubated with 60% glucose at 50° C. for66 h with a (Glc-Gal)/Gal ratio of 3.39 and 4.09, respectively. Whereasa smaller increase in (Glc-Gal)/Gal ratio is seen for Kluyveromyceslactis β-galactosidase (GH2_6). This suggests that for subfamily 5 ofthe glycosyl hydrolase family 2 (GH2_5), glycation has a more pronouncedaffect to shift the enzyme molecule from having a hydrolytic to atransferase activity, than for subfamily 6 of GH2 (GH2_6).

All control samples BBB-1-C, K. lactis and B. circulans have a(Glc-Gal)/Gal ratio close to zero. When compared to the small butpositive values for the controls of Example 5, this is as expected asthere is only ˜5% lactose in milk whereas the controls in Table 5 wereincubated at 60% lactose (a high lactose conc. favours transferaseactivity).

Glycation in lower glucose conc. and lower temperatures could also beachieved at prolonged incubation times as seen in Table 6, where BBB-3incubated in 40% glucose at 40° C. for 672 h resulted in a (Glc-Gal)/Galratio of 0.75 compared to control which has a value of zero. Anadditional control was made incubating Bifidobacterium bifidumβ-galactosidase in 40% glycerol at 40° C. for 672 h which has nodetectable effect on the (Glc-Gal)/Gal ratio as a value of zero wasobtained just as for the control. This additional experiment confirmsthat glycation and not heat-treatment as such is responsible fortransforming the enzyme from a hydrolytic enzyme to a more transferringenzyme.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. A formulation comprising a polypeptide having beta-galactosidaseactivity and at least 30 wt % of a reducing sugar, preferably fructose,galactose, glucose, or lactose; wherein the polypeptide havingbeta-galactosidase activity has been modified by glycation of at leastone lysine and/or arginine residue.
 2. The formulation of claim 1,wherein the polypeptide having beta-galactosidase activity has beenmodified by glycation of at least 1%, preferably at least 3%, morepreferably at least 5%, of the lysine and arginine residues of thepolypeptide.
 3. The formulation of claim 1 having an activity of200-20,000 LAU(C)/g.
 4. The formulation of claim 1 which is a liquidformulation and which preferably has an activity of 200-15,000 LAU(C)/g.5. The formulation of claim 1, comprising 40-65 wt % sugar, wherein thesugar is preferably glucose.
 6. The formulation of claim 1, which issubstantially free of glycerol, and which optionally further comprisessodium chloride or potassium chloride, preferably in the range of 0.01-5wt %. 7-9. (canceled)
 10. A method of modifying by glycation apolypeptide having beta-galactosidase activity comprising contacting thepolypeptide with 30-90 wt % of a reducing sugar for a time andtemperature sufficient to produce a polypeptide modified by glycation.11. The method of claim 10, wherein the polypeptide havingbeta-galactosidase activity modified by glycation has improvedtransgalactosylating activity as compared to the polypeptide havingbeta-galactosidase activity which has not been modified by glycation.12. The method of claim 10, wherein the polypeptide havingbeta-galactosidase activity is modified by glycation of at least 1% ofthe lysine and arginine residues of the polypeptide.
 13. The method ofclaim 10, comprising contacting the polypeptide havingbeta-galactosidase activity at pH 5-8 for a time of 3-100 hours at atemperature of 50-80° C.
 14. (canceled)
 15. A method for producinggalacto-oligosaccharides (GOS) comprising contacting the formulation ofclaim 1 with lactose.